Heterotrimeric (alpha-beta-gamma) Gs mediates agonist-induced stimulation of adenylyl cyclase (AC). The alpha subunit (Gs-alpha) has intrinsic GTPase activity. Gs is activated when Gs-alpha binds GTP or a GTP analog (e.g. GTPgammaS) and it is inactivated when GTP is hydrolyzed. Gs-alpha is composed of two domains, one a ras-like GTPase domain and the other a predominately alpha helical domain, whose function remains largely unclear. We have found through mutagenesis that a non-conserved arginine in a region of the ras-like domain known as switch 3 forms a bond with a conserved glutamine in the helical domain. These two amino acids create a "lid' over the active site providing direct evidence that interaction between the two domains is important for high affinity, guanine nucleotide binding, and consequently the stability of Gs-alpha. We have also demonstrated by mutagenesis that a strictly conserved glutamate in switch 3 is important in the activation mechanism because it forms contacts with a region known as switch 2, thereby permitting efficient binding of the transition state that occurs when GTP is hydrolyzed. Under certain conditions the activation of Gs in solution can be accompanied by dissociation of Gs-alpha from the G protein beta-gamma subunit complex (G-beta-gamma). Nevertheless, we have produced compelling evidence that subunit dissociation does not necessarily occur when Gs is activated in solution. We are continuing these investigations by studying the kinetics of G protein subunit dissociation and association under different conditions using surface plasmon resonance spectroscopy. However, what happens when membrane bound Gs is activated is the more physiologically relevant as well as the more difficult question to answer, and this has been a focus of current research. Cholera toxin (CTx) activates AC by ADP-ribosylating Gs-alpha. In solution Gs-alpha is a substrate for CTx only when it is associated with G-beta-gamma. To determine if the substrate specificity for CTx is the same when Gs-alpha is membrane bound, Gs-alpha deficient cyc- membranes were "striped" of G-beta-gamma (cyc-/-) with a solution containing the detergent CHAPS. When Gs-alpha was incorporated into cyc-/-, it was a substrate for CTx only when G-beta-gamma was also incorporated indicating that the Gs heterotrimer but not free Gs-alpha is the substrate for CTx in cell membranes. The stimulation of AC by GTPgammaS-activated Gs-alpha in cyc-/- was also dependent upon the simultaneous incorporation of G-beta-gamma. Furthermore, there was a correlation between the dose dependent effects of G-beta-gamma on the stimulation of AC by GTPgammaS-activated Gs-alpha and the CTx catalyzed ADP-ribosylation of GTPgammaS-liganded Gs-alpha in cyc-/-. These data suggest that AC is stimulated by an activated Gs heterotrimer in cell membranes.